Protein-protein interfaces are mostly hydrophobic. Nevertheless, there are some critical residues at specific locations in these binding sites, possibly serving as hot spots. Previous computational studies have shown that there is a correspondence between the experimentally found energy hot spots and structurally conserved residues. Recently, we have extracted a non-redundant dataset of protein-protein interfaces from the PDB. The interfaces are clustered based on their spatial structural similarities,regardless of the connectivity of their residues on the protein chains. We have obtained 103 clusters which have at least five non-homologous membersfrom this dataset. These serve as a rich source of data for analysis of protein interfaces. In this study, we have extracted all hot spots in these 103 interface clusters and compared them with the alanine scanning mutagenesis dataset. We have also studied how these hot spots change with biological functions, interface sizes, and residue types. Results show that there are differences among different sets of proteins. These may further aid in distinguishing between binding sites and the remainder of the protein surfaces.Shape complementarity is thought to be the basis of protein-protein interactions, but there is a significant number of interfacial pockets that are not filled by protein atoms. We study the shape properties ofinterfacial pockets in protein-protein interactions in a large, diverse, recently derived structurally nonredundant protein interface dataset. Pockets in interfacial regions of monomers are classified into two types: unfilled pockets and complemented pockets. Unfilled pockets remain unfilled after complexation, whereas complemented pockets are filled by the binding partner. Unfilled pockets are widely distributed in protein-protein interfaces, suggesting that shape complementarity is far from perfect. About one-third protein-protein interfaces contain at leastone complemented pocket. We further examine the interface details. Experimentally, a small number of residues are hot spots, accounting for a large portion of the total binding free energy. Computationally, a set ofstructurally conserved residues are observed at the interfaces, correlating with the hot spots. We found that structurally conserved residues are significantly favored to be located in complemented pockets, and significantly unfavored to be in unfilled pockets. Similar patterns are found for experimental hot spots obtained from Thorn and Bogan's ASEdb database. Among the 8 protein-protein complexes where residues on both sides have been mutated, three complexes have complemented pockets in the interfaces. Characterization of complemented pockets identifies about 61% (19 out of 31) of all hot spots. 85% (11 out of 13) of the hot spots with > 4 kcal/mol binding energy change contained in the three protein-protein complexes. Our results suggest that complemented pockets and corresponding protruding residues are among the important features that determine protein-protein interactions.Structurally conserved residues at protein-protein interfaces correlate with the experimental alanine-scanning hot spots. Here, we investigate the organization of these conserved, computational hot spots and their contribution to the stability of protein associations. We find that computational hot spots are not homogeneously distributed along the protein interfaces; rather they are clustered within locally tightly packed regions. Within the dense clusters, they form a network of interactions and consequently their contributions to the stability of the complex are cooperative; however the contributions of independent clusters are additive. This suggests that the binding free energy is not a simple summation of the single hot spot residue contributions. As expected, around the hot spot residues we observe moderately conserved residues, further highlighting the crucial role of the conserved interactions in the local densely packed environment. The conserved occurrence of these organizations suggests that they are advantageous for protein-protein associations. Interestingly, the total number of hydrogen bonds and salt bridges contributed by hot spots is as expected. Thus, H-bond forming residues may use a "hot spot for water exclusion" mechanism. Since conserved residues are located within highly packed regions, water molecules are easily removed upon binding, strengthening electrostatic contributions of charge-charge interactions. Hence, the picture that emerges is that protein-protein associations are optimized locally, with the clustered, networked, highly packed structurally conserved residues contributing dominantly and cooperatively to the stability of the complex. When addressing the crucial question of "what are the preferred ways of proteins to associate", these findings point toward a critical involvement of hot regions in protein-protein interactions.